Modified protein encoding sequences having increased rare hexamer content

Abstract

This invention provides a modified protein encoding sequence containing nucleotide substitutions at multiple locations in the protein encoding sequence, wherein the substitutions introduce rare hexamers. These hexamers may be Frame Dependent, or depleted in only the reading frame, or Frame Independent, or depleted in all three frames. Modified protein encoding se quences of the present invention may include modified viruses useful for vaccines.

1 . A modified protein encoding sequence comprising a polynucleotide sequence derived from a target protein encoding sequence, wherein the modified protein encoding sequence encodes a polypeptide having substantially the same amino acid sequence as the polypeptide encoded by the target protein encoding sequence and comprises a plurality of additional hexamers selected from one or more of the group consisting of SEQ ID NO:19 to SEQ ID NO:418 compared to the target protein encoding sequence. 
     
     
         2 . The modified protein encoding sequence of  claim 1 , wherein the plurality of hexamers comprises frame independent hexamers. 
     
     
         3 . The modified protein encoding sequence of  claim 1 , wherein the plurality of hexamers comprises frame dependent hexamers. 
     
     
         4 . The modified protein encoding sequence of  claim 1 , wherein the modified protein encoding sequence comprises more than about 50 additional hexamers selected from one or more of the group consisting of SEQ ID NO:19 to SEQ ID NO:418 compared to the target protein encoding sequence. 
     
     
         5 - 9 . (canceled) 
     
     
         10 . The modified protein encoding sequence of  claim 1 , wherein the modified protein encoding sequence has reduced expression in mammalian cells compared to the unmodified protein encoding sequence. 
     
     
         11 - 12 . (canceled) 
     
     
         13 . The modified protein encoding sequence of  claim 1 , wherein the hexamers are introduced by rearranging synonymous codons of the target protein encoding sequence. 
     
     
         14 . The modified protein encoding sequence of  claim 1 , wherein the hexamers are introduced by substituting synonymous codons of the target protein encoding sequence. 
     
     
         15 . The modified protein encoding sequence of  claim 1 , wherein the target protein encoding sequence encodes a viral protein. 
     
     
         16 . A modified virus comprising the modified protein encoding sequence of  claim 15 . 
     
     
         17 . A method for reducing the expression of a target protein comprising introducing into the target protein encoding sequence a plurality of hexamers selected from one or more of the group consisting of SEQ ID NO: 19 to SEQ ID NO: 418 without altering the polypeptide sequence encoded by the target protein encoding sequence. 
     
     
         18 . The method of  claim 17 , wherein the plurality of hexamers comprises frame dependent hexamers. 
     
     
         19 . The method of  claim 17 , wherein the plurality of hexamers comprises frame independent hexamers. 
     
     
         20 . The method of  claim 17 , wherein greater than about 50 hexamers are introduced into the target protein encoding sequence. 
     
     
         21 . (canceled) 
     
     
         22 . The method of  claim 17 , wherein hexamers are introduced by rearranging synonymous codons. 
     
     
         23 . The method of  claim 17 , wherein hexamers are introduced by substituting synonymous codons. 
     
     
         24 . The method of  claim 17 , wherein the target protein encoding sequence is a viral gene. 
     
     
         25 . A modified protein encoding sequence comprising a polynucleotide sequence derived from a target protein encoding sequence, wherein the modified protein encoding sequence encodes a polypeptide having substantially the same amino acid sequence as the polypeptide encoded by the target protein encoding sequence and comprises at least one of: a plurality of frame dependent hexamers each having a frame dependent score less than about −0.51 and a plurality of frame independent hexamers each having a frame independent score less than about −0.33. 
     
     
         26 - 28 . (canceled) 
     
     
         29 . The modified protein encoding sequence of  claim 25 , wherein the modified protein encoding sequence comprises more than about 50 additional hexamers selected from one or more of the group consisting SEQ ID NO:19 to SEQ ID NO:418 compared to the target protein encoding sequence. 
     
     
         30 - 35 . (canceled) 
     
     
         36 . The modified protein encoding sequence of  claim 25 , wherein the modified protein encoding sequence has reduced expression in mammalian cells compared to the unmodified protein encoding sequence. 37-38. (cancelled). 
     
     
         39 . The modified protein encoding sequence of  claim 25 , wherein the hexamers are introduced by rearranging synonymous codons of the target protein encoding sequence. 
     
     
         40 . The modified protein encoding sequence of  claim 25 , wherein the hexamers are introduced by substituting synonymous codons of the target protein encoding sequence. 
     
     
         41 . The modified protein encoding sequence of  claim 25 , wherein the target protein encoding sequence encodes a viral protein.